Non-equilibrium folding of individual DNA molecules recaptured up to 1000 times in a solid state nanopore

We investigate translocation of linear and circular double-stranded DNA molecules through solid state nanopores where each molecule is recaptured and re-translocated many times. Single molecules can be recaptured by switching voltage polarity for hundreds or even thousands of times. The large number of recapture events allows statistics on the translocation of individual molecules. Surprisingly, we observe that recaptured DNA molecules do not translocate in a linear head-to-tail fashion, but instead translocate as a folded blob where multiple parts of the DNA molecule simultaneously translocate through the pore in parallel. This folding is observed through the presence of up to 13 DNA double strands from the same molecule simultaneously inside the pore, as well as many smaller fold numbers occurring during the course of a translocation event. The strong folding is particularly prominent when the molecule is recaptured at short timescales, i.e. shorter than its characteristic time to relax to its equilibrium configuration. At longer recapture times, both the amount of folding and the mean duration of translocation approach the values observed in non-recapture experiments. The data shows that the translocation time of a molecule depends on the molecule's conformation at the start of the translocation process, with extended molecules having a longer translocation time. The observations can be attributed to a high-density non-equilibrium DNA configuration that arises in the close vicinity of the nanopore immediately after translocation, which dissipates on a timescale given by the Zimm relaxation time.